scholarly journals Cloning, Sequencing, and Phenotypic Characterization of the rpoS Gene from Pseudomonas putida KT2440

1998 ◽  
Vol 180 (13) ◽  
pp. 3421-3431 ◽  
Author(s):  
María Isabel Ramos-González ◽  
Søren Molin
Keyword(s):  
Pseudomonas Putida ◽  
Carbon Starvation ◽  
Phenotypic Characterization ◽  
Gram Negative Bacteria ◽  
Pseudomonas Putida Kt2440 ◽  
Gram Negative ◽  
Acid Sensitivity ◽  
E Coli ◽  
Short Period ◽  
In Cells

ABSTRACT A gene homologous to the rpoS gene of Escherichia coli was cloned from a Pseudomonas putida KT2440 gene bank by complementation of the rpoS-deficient strainE. coli ZK918. The rpoS gene of P. putida complemented the acid sensitivity and catalase deficiency of the rpoS mutant of E. coli and stimulated expression of the RpoS-controlled promoter,bolAp 1. The gene was sequenced and found to be highly similar to the rpoS genes of other gram-negative bacteria. Like in other gram-negative bacteria, a homolog of thenlpD gene was found upstream to the rpoS gene. A transcriptional fusion of the promoter of the P. putida rpoS gene to the luxAB genes from Vibrio harveyi was constructed and used as an inactivated allele ofrpoS for gene replacement of the wild-type copy in the chromosome of P. putida. The resultantrpoS mutant of P. putida, C1R1, showed reduced survival of carbon starvation and reduced cross-protection against other types of stress in cells starved for carbon, in particular after a challenge with ethanol. Survival in soil amended with m-methylbenzoate was also reduced in the mutant strain P. putida C1R1. The RpoS protein ofP. putida controls the expression of more than 50 peptides, which are normally expressed in cells after a short period of carbon starvation.

scholarly journals Adaptation of the Yeast URA3 Selection System to Gram-Negative Bacteria and Generation of a ΔbetCDE Pseudomonas putida Strain

2005 ◽  
Vol 71 (2) ◽  
pp. 883-892 ◽  
Author(s):  
Teca Calcagno Galvão ◽  
Víctor de Lorenzo
Keyword(s):  
Pseudomonas Putida ◽  
General Procedure ◽  
Gene Replacement ◽  
Selection Marker ◽  
Homologous Gene ◽  
Gram Negative Bacteria ◽  
Selection System ◽  
Gram Negative ◽  
Acid Sensitivity ◽  
Delivery Vector

ABSTRACT A general procedure for efficient generation of gene knockouts in gram-negative bacteria by the adaptation of the Saccharomyces cerevisiae URA3 selection system is described. A Pseudomonas putida strain lacking the URA3 homolog pyrF (encoding orotidine-5′-phosphate decarboxylase) was constructed, allowing the use of a plasmid-borne copy of the gene as the target of selection. The delivery vector pTEC contains the pyrF gene and promoter, a conditional origin of replication (oriR6K), an origin of transfer (mobRK2), and an antibiotic selection marker flanked by multiple sites for cloning appropriate DNA segments. The versatility of pyrF as a selection system, allowing both positive and negative selection of the marker, and the robustness of the selection, where pyrF is associated with uracil prototrophy and fluoroorotic acid sensitivity, make this setup a powerful tool for efficient homologous gene replacement in gram-negative bacteria. The system has been instrumental for complete deletion of the P. putida choline-O-sulfate utilization operon betCDE, a mutant which could not be produced by any of the other genetic strategies available.

Disinfection of Fruits with Activated Water

2019 ◽  
Vol 10 ◽  
pp. 1864-1872
Author(s):  
Prof. Teodora P. Popova
Keyword(s):  
Antimicrobial Activity ◽  
Aqueous Solutions ◽  
Antimicrobial Properties ◽  
The Other ◽  
Gram Negative Bacteria ◽  
High Antimicrobial Activity ◽  
Gram Negative ◽  
E Coli ◽  
Number Of Microorganisms ◽  
Lower Effect

The effect of ionized aqueous solutions (anolytes and catholyte) in the processing of fruits (cherries, morellos, and strawberries) for decontamination has been tested. Freshly prepared analytes and catholyte without the addition of salts were used, as well as stored for 7 months anolytes, prepared with 0.5% NaCl and a combination of 0.5% NaCl and 0.5% Na2CO3. The anolyte prepared with a combination of 0.5% NaCl and 0.5% Na2CO3, as well as the anolyte obtained with 0.5% NaCl, exhibit high antimicrobial activity against the surface microflora of strawberries, cherries, and sour cherries. They inactivate E. coli for 15 minutes. The other species of the fam. Enterobacteriaceae were also affected to the maximum extent, as is the total number of microorganisms, especially in cherries and sour cherries. Even stored for 7 months, they largely retain their antimicrobial properties. Anolyte and catholyte, obtained without the addition of salts, showed a lower effect on the total number of microorganisms, but had a significant effect on Gram-negative bacteria, and especially with regard to the sanitary indicative E. coli.

Structure-Activity Relationship and Antimicrobial Evaluation of N-Phenylpyrazole Curcumin Derivatives

2020 ◽  
Vol 16 (4) ◽  
pp. 481-488
Author(s):  
Heli Sanghvi ◽  
Satyendra Mishra
Keyword(s):  
Antibacterial Activity ◽  
Gram Negative Bacteria ◽  
Pyrazole Derivatives ◽  
Gram Positive ◽  
Gram Negative ◽  
E Coli ◽  
Curcumin Derivatives ◽  
Structure Activity ◽  
Electron Donating Groups ◽  
Derivatives Of

Background: Curcumin, one of the most important pharmacologically significant natural products, has gained significant consideration among scientists for decades since its multipharmacological activities. 1, 3-Dicarbonyl moiety of curcumin was found to be accountable for the rapid degradation of curcumin molecule. The aim of present work is to replace 1, 3-dicarbonyl moiety of curcumin by pyrazole and phenylpyrazole derivatives with a view to improving its stability and to investigate the role of substitution in N-phenylpyrazole curcumin on its antibacterial activity against both Gram-positive as well as Gram-negative bacteria. Methods: Pyrazole derivatives of curcumin were prepared by heating curcumin with phenyhydrazine/ substituted phenyhydrazine derivatives in AcOH. The residue was purified by silica gel column chromatography. Structures of purified compounds were confirmed by 1H NMR and Mass spectroscopy. The synthesized compounds were evaluated for their antibacterial activity by the microdilution broth susceptibility test method against gram positive (S. aureus) and gram negative (E. coli). Results: Effects of substitution in N-phenylpyrazole curcumin derivatives against S. aureus and E. coli were studied. The most active N-(3-Nitrophenylpyrazole) curcumin (12) exhibits twenty-fold more potency against S. aureus (MIC: 10μg/mL)) and N-(2-Fluoroophenylpyrazole) curcumin (5) fivefold more potency against E. coli (MIC; 50 μg/mL) than N-phenylpyrazole curcumin (4). Whereas, a remarkable decline in anti-bacterial activity against S. aureus and E. coli was observed when electron donating groups were incorporated in N-phenylpyrazole curcumin (4). Comparative studies of synthesized compounds suggest the effects of electron withdrawing and electron donating groups on unsubstituted phenylpyrazole curcumin (4). Conclusion: The structure-activity relationship (SAR) results indicated that the electron withdrawing and electron donating at N-phenylpyrazole curcumin played key roles for their bacterial inhibitory effects. The results of the antibacterial evaluation showed that the synthesized pyrazole derivatives of curcumin displayed moderate to very high activity in S. aureus. In conclusion, the series of novel curcumin derivatives were designed, synthesized and tested for their antibacterial activities against S. aureus and E. coli. Among them, N-(3-Nitrophenylpyrazole curcumin; 12) was most active against S. aureus (Gram-positive) and N-(2-Fluoroophenylpyrazole) curcumin (5) against E. coli (Gram-negative) bacteria.

Quality Improvement of the DNA extracted by boiling method in Gram negative bacteria

2017 ◽  
Vol 6 (04) ◽  
pp. 5347 ◽  
Author(s):  
Omar B. Ahmed* ◽  
Anas S. Dablool
Keyword(s):  
Dna Extraction ◽  
Control Method ◽  
Extraction Procedure ◽  
Cost Effective ◽  
High Yield ◽  
Gram Negative Bacteria ◽  
Bacterial Dna ◽  
Gram Negative ◽  
E Coli

Several methods of Deoxyribonucleic acid (DNA) extraction have been applied to extract bacterial DNA. The amount and the quality of the DNA obtained for each one of those methods are variable. The study aimed to evaluate bacterial DNA extraction using conventional boiling method followed by alcohol precipitation. DNA extraction from Gram negative bacilli was extracted and precipitated using boiling method with further precipitation by ethanol. The extraction procedure performed using the boiling method resulted in high DNA yields for both E. coli and K. pneumoniae bacteria in (199.7 and 285.7μg/ml, respectively) which was close to control method (229.3 and 440.3μg/ml). It was concluded that after alcohol precipitation boiling procedure was easy, cost-effective, and applicable for high-yield quality of DNA in Gram-negative bacteria.

scholarly journals Activity of Specialized Biomolecules against Gram-Positive and Gram-Negative Bacteria

Antibiotics ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 314 ◽  
Author(s):  
Tânia D. Tavares ◽  
Joana C. Antunes ◽  
Jorge Padrão ◽  
Ana I. Ribeiro ◽  
Andrea Zille ◽  
...  
Keyword(s):  
Cell Morphology ◽  
Time Averaging ◽  
Gram Negative Bacteria ◽  
Gram Positive ◽  
Intracellular Space ◽  
Gram Negative ◽  
Tea Tree ◽  
Minimal Inhibitory Concentrations ◽  
Agar Diffusion Test ◽  
Short Period

The increased resistance of bacteria against conventional pharmaceutical solutions, the antibiotics, has raised serious health concerns. This has stimulated interest in the development of bio-based therapeutics with limited resistance, namely, essential oils (EOs) or antimicrobial peptides (AMPs). This study envisaged the evaluation of the antimicrobial efficacy of selected biomolecules, namely LL37, pexiganan, tea tree oil (TTO), cinnamon leaf oil (CLO) and niaouli oil (NO), against four bacteria commonly associated to nosocomial infections: Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa. The antibiotic vancomycin and silver nanoparticles (AgNPs) were used as control compounds for comparison purposes. The biomolecules were initially screened for their antibacterial efficacy using the agar-diffusion test, followed by the determination of minimal inhibitory concentrations (MICs), kill-time kinetics and the evaluation of the cell morphology upon 24 h exposure. All agents were effective against the selected bacteria. Interestingly, the AgNPs required a higher concentration (4000–1250 μg/mL) to induce the same effects as the AMPs (500–7.8 μg/mL) or EOs (365.2–19.7 μg/mL). Pexiganan and CLO were the most effective biomolecules, requiring lower concentrations to kill both Gram-positive and Gram-negative bacteria (62.5–7.8 μg/mL and 39.3–19.7 μg/mL, respectively), within a short period of time (averaging 2 h 15 min for all bacteria). Most biomolecules apparently disrupted the bacteria membrane stability due to the observed cell morphology deformation and by effecting on the intracellular space. AMPs were observed to induce morphological deformations and cellular content release, while EOs were seen to split and completely envelope bacteria. Data unraveled more of the potential of these new biomolecules as replacements for the conventional antibiotics and allowed us to take a step forward in the understanding of their mechanisms of action against infection-related bacteria.

scholarly journals Clonal Clusters, Molecular Resistance Mechanisms and Virulence Factors of Gram-Negative Bacteria Isolated from Chronic Wounds in Ghana

Antibiotics ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 339
Author(s):  
Denise Dekker ◽  
Frederik Pankok ◽  
Thorsten Thye ◽  
Stefan Taudien ◽  
Kwabena Oppong ◽  
...  
Keyword(s):  
Molecular Epidemiology ◽  
Virulence Factors ◽  
Iron Uptake ◽  
Chronic Wounds ◽  
Sub Saharan Africa ◽  
Gram Negative Bacteria ◽  
Beta Lactamase ◽  
Secretion Systems ◽  
Gram Negative ◽  
E Coli

Wound infections are common medical problems in sub-Saharan Africa but data on the molecular epidemiology are rare. Within this study we assessed the clonal lineages, resistance genes and virulence factors of Gram-negative bacteria isolated from Ghanaian patients with chronic wounds. From a previous study, 49 Pseudomonas aeruginosa, 21 Klebsiellapneumoniae complex members and 12 Escherichia coli were subjected to whole genome sequencing. Sequence analysis indicated high clonal diversity with only nine P. aeruginosa clusters comprising two strains each and one E. coli cluster comprising three strains with high phylogenetic relationship suggesting nosocomial transmission. Acquired beta-lactamase genes were observed in some isolates next to a broad spectrum of additional genetic resistance determinants. Phenotypical expression of extended-spectrum beta-lactamase activity in the Enterobacterales was associated with blaCTX-M-15 genes, which are frequent in Ghana. Frequently recorded virulence genes comprised genes related to invasion and iron-uptake in E. coli, genes related to adherence, iron-uptake, secretion systems and antiphagocytosis in P. aeruginosa and genes related to adherence, biofilm formation, immune evasion, iron-uptake and secretion systems in K. pneumonia complex. In summary, the study provides a piece in the puzzle of the molecular epidemiology of Gram-negative bacteria in chronic wounds in rural Ghana.

scholarly journals Apomorphine Targets the Pleiotropic Bacterial Regulator Hfq

Antibiotics ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 257
Author(s):  
Florian Turbant ◽  
David Partouche ◽  
Omar El Hamoui ◽  
Sylvain Trépout ◽  
Théa Legoubey ◽  
...  
Keyword(s):  
Small Rnas ◽  
Antibacterial Agents ◽  
Amyloid Fibrils ◽  
Noncoding Rnas ◽  
Amyloid Formation ◽  
Gram Negative Bacteria ◽  
Translation Efficiency ◽  
Gram Negative ◽  
Bacterial Adaptation ◽  
E Coli

Hfq is a bacterial regulator with key roles in gene expression. The protein notably regulates translation efficiency and RNA decay in Gram-negative bacteria, thanks to its binding to small regulatory noncoding RNAs. This property is of primary importance for bacterial adaptation and survival in hosts. Small RNAs and Hfq are, for instance, involved in the response to antibiotics. Previous work has shown that the E. coli Hfq C-terminal region (Hfq-CTR) self-assembles into an amyloid structure. It was also demonstrated that the green tea compound EpiGallo Catechin Gallate (EGCG) binds to Hfq-CTR amyloid fibrils and remodels them into nonamyloid structures. Thus, compounds that target the amyloid region of Hfq may be used as antibacterial agents. Here, we show that another compound that inhibits amyloid formation, apomorphine, may also serve as a new antibacterial. Our results provide an alternative in order to repurpose apomorphine, commonly used in the treatment of Parkinson’s disease, as an antibiotic to block bacterial adaptation to treat infections.

scholarly journals Proteomic response of Escherichia coli to a membrane lytic and iron chelating truncated Amaranthus tricolor defensin

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Tessa B. Moyer ◽  
Ashleigh L. Purvis ◽  
Andrew J. Wommack ◽  
Leslie M. Hicks
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Antimicrobial Peptides ◽  
Mechanism Of Action ◽  
Gram Negative Bacteria ◽  
Amaranthus Tricolor ◽  
Core Motif ◽  
Gram Negative ◽  
E Coli ◽  
Membrane Lysis

Abstract Background Plant defensins are a broadly distributed family of antimicrobial peptides which have been primarily studied for agriculturally relevant antifungal activity. Recent studies have probed defensins against Gram-negative bacteria revealing evidence for multiple mechanisms of action including membrane lysis and ribosomal inhibition. Herein, a truncated synthetic analog containing the γ-core motif of Amaranthus tricolor DEF2 (Atr-DEF2) reveals Gram-negative antibacterial activity and its mechanism of action is probed via proteomics, outer membrane permeability studies, and iron reduction/chelation assays. Results Atr-DEF2(G39-C54) demonstrated activity against two Gram-negative human bacterial pathogens, Escherichia coli and Klebsiella pneumoniae. Quantitative proteomics revealed changes in the E. coli proteome in response to treatment of sub-lethal concentrations of the truncated defensin, including bacterial outer membrane (OM) and iron acquisition/processing related proteins. Modification of OM charge is a common response of Gram-negative bacteria to membrane lytic antimicrobial peptides (AMPs) to reduce electrostatic interactions, and this mechanism of action was confirmed for Atr-DEF2(G39-C54) via an N-phenylnaphthalen-1-amine uptake assay. Additionally, in vitro assays confirmed the capacity of Atr-DEF2(G39-C54) to reduce Fe3+ and chelate Fe2+ at cell culture relevant concentrations, thus limiting the availability of essential enzymatic cofactors. Conclusions This study highlights the utility of plant defensin γ-core motif synthetic analogs for characterization of novel defensin activity. Proteomic changes in E. coli after treatment with Atr-DEF2(G39-C54) supported the hypothesis that membrane lysis is an important component of γ-core motif mediated antibacterial activity but also emphasized that other properties, such as metal sequestration, may contribute to a multifaceted mechanism of action.

scholarly journals An Application of Imipenem Discs or P. aeruginosa ATCC 27853 Reference Strain Increases Sensitivity of Carbapenem Inactivation Method for Non-Fermenting Gram-Negative Bacteria

Antibiotics ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 875
Author(s):  
Tomasz Bogiel ◽  
Mateusz Rzepka ◽  
Eugenia Gospodarek-Komkowska
Keyword(s):  
Human Population ◽  
Reference Strain ◽  
Resistance Mechanisms ◽  
Gram Negative Bacteria ◽  
Opportunistic Pathogens ◽  
Beta Lactams ◽  
Gram Negative ◽  
E Coli ◽  
Human Infections ◽  
Reliable Diagnostic Method

Non-fermenting Gram-negative rods are one of the most commonly isolated bacteria from human infections. These microorganisms are typically opportunistic pathogens that pose a serious threat to public health due to possibility of transmission in the human population. Resistance to beta-lactams, due to carbapenemases synthesis, is one of the most important antimicrobial resistance mechanisms amongst them. The aim of this study was to evaluate the usefulness of the Carbapenem Inactivation Method (CIM), and its modifications, for the detection of carbapenemase activity amongst non-fermenting Gram-negative rods. This research involved 81 strains of Gram-negative rods. Of the tested strains, 55 (67.9%) synthesized carbapenemases. For non-fermenting rods, 100% sensitivity and specificity was obtained in the version of the CIM test using imipenem discs and E. coli ATCC 25922 strain. The CIM test allows for differentiation of carbapenems resistance mechanisms resulting from carbapenemase synthesis from other resistance types. It is a reliable diagnostic method for the detection of carbapenemase activity amongst non-fermenting Gram-negative rods. Application of imipenem discs and P. aeruginosa ATCC 27853 reference strain increases CIM results sensitivity, while imipenem discs and E. coli ATCC 25922 strain use maintains full precision of the test for non-fermenting rods.

Sign in / Sign up

Export Citation Format

Share Document